Method for selectively separating blood cells by using lectin

ABSTRACT

The present invention provides a method for selective and high-yield separation, concentration, and recovery of desired cells from a blood sample. The method of the present invention is characterized in that the blood sample is caused to interact with lectins under conditions in which the cell membranes are inactive and cell-lectin complexes/non-complexes are formed, the sample containing these cell-lectin complexes/non-complexes is incubated together with a substrate, the surface of which is covered with polymers having carbohydrate chains which are specifically recognized by the lectins, and the cells are immobilized on the surface of the substrate via the lectins, and subsequently, the liquid layer and the solid phase are separated, and the desired blood cells are recovered from the liquid phase and/or the solid phase, and these lectins are in such an amount that although the cells to be recovered from the solid phase bind to the solid phase with the polymer, the cells to be recovered from the liquid phase do not bind to the solid phase with the polymer.

FIELD OF THE INVENTION

The present invention relates to a method for the separation of bloodcells using lectins, and in particular, relates to a method for theselective separation and recovery of desired blood cells, via lectin,from a sample containing both mature cells and immature cells containedin peripheral blood, bone marrow fluid, umbilical cord blood, or thelike.

BACKGROUND OF THE INVENTION

Hematopoietic stem cells are cells which combine the potential ofmulti-differentiation and autoreproduction. The potential ofautoreproduction is most important for hematopoiesis in order that bloodcells not become exhausted over the course of a lifetime. With respectto the ability of the hematopoietic stem cells to multi-differentiate,as shown in FIG. 1, stem cells differentiate into myeloid stem cells andlymphatic stem cells, and these further differentiate into platelets,(mature) erythrocytes, granulocytes, monocytes, or the like from themyeloid stem cells, while blood cells such as T cells or B cells or thelike are produced from the lymphatic stem cells.

Blood cells have a lifetime and are consumed in accordance with avariety of physiological needs, so that it is necessary that the bloodcells be appropriately replenished by differentiation from stem cells.In patients suffering from, for example, acute myelogenous leukemia,there are irregularities in the differentiated functional blood cellsthemselves, as well as in the stem cell differentiation, so that thereplenishment of functional red blood cells, white blood cells,platelets and the like is difficult. The transplantation ofhematopoietic stem cells offers a treatment method for such blooddiseases which does not have the side effects of chemotherapy andresults in the recovery of functional hematopoiesis through thedifferentiation and regeneration of these cells. However, despite theseadvantages, the acquisition of stem cells is difficult, as they arealmost all distributed in the bone marrow. Although placental blood andumbilical cord blood are comparatively enriched in stem cells, so that aless invasive method of obtaining them is possible, they can be usedonly in childbirth. On the other hand, in the peripheral blood which canbe obtained from a donor in the least invasive way, the amounts of stemcells are further reduced which makes the peripheral blood lesspractical.

Furthermore, in transplanted blood cells containing stem cells,immunological rejection reaction, termed graft-versus-host disease(GVHD), may be induced when the HLA type of the patient and donor do notmatch. Accordingly, in order to conduct effective and safetransplantation of stem cells, it is necessary to obtain a stem cellsample from which lymphocyte fractions which give rise to GVHD areremoved.

Furthermore, if pure stem cells could be isolated, they could beeffectively stimulated and expanded using cytokines. Consequently, stemcell isolation could contribute to the development of stem cell banks inwhich such cells were stored for later use.

On the other hand, in concert with the recent development in geneticmanipulation techniques, efforts have been made to conduct prenatal genediagnosis for fetal nucleated cells. Fetal nucleated cells fordiagnostic use which are currently clinically employed, are collectedthrough invasive methods such as amniocentesis, chorionic villoussampling, and fetal blood collection, and these carry the risk ofinfection and amniorrhexis. It is conventionally known that fetal cellsare admixed in the maternal blood, and the use of maternal peripheralblood to obtain fetal nucleated cells as a non-invasive collection hasbeen considered; however, nucleated red blood cells (NRBC) being likelyfetal cells are contained in the maternal peripheral blood in very smallamounts, being only 1 in 10⁵-10⁷ of the total nucleated cells in theperipheral blood, so that the key to genetic diagnosis of fetal cellshas been how to concentrate, separate, or identify such cells.

In addition, it is known that gene diagnosis is effective for thetherapy of leukemia For example, since leukemia includes various typessuch as myeloid type in which hematopoietic cells themselves arepathologic or other type in which peripheral lymphocytes or monocytesbecome malignant, it is necessary to identify the type of leukemia fordetermining optimal dosing or therapeutic regimen. Moreover, geneticexamination is needed to know the stage of differentiation of bloodcells in which a carcinogenic factor is induced because the detection ofthe stage in which carcinogenic cells occur would contribute not only tothe treatment of leukemia but also to clinically important matters suchas prevention or recurrence of cancer. In such an examination ofleukemia, it is also necessary to simplify and make effective the genediagnosis in each differentiation stage by selecting and purifyingimmature hematopoietic blood cells and proliferating and differentiatingthem with cytokines.

The present inventors have conducted research which focused on thespecific interactions between carbohydrates and other biologicalsubstances, and have filed a patent application on a method forselectively binding lectins, carbohydrate-specific proteins, to a solidsupport, such as a dish or the like, covered with syntheticglycoconjugate polymers including carbohydrate moieties (Japanese PatentApplication No. Hei 8-59695).

On the other hand, as is shown in FIG. 1, hematocytes derived fromhematopoietic stem cells express a variety of carbohydrate chains on thecell surface in accordance with the maturation thereof. In FIG. 1, thedesignation “Gal” indicates galactose, “Glu” indicates glucose, and“Lac” indicates lactose (Glu-Gal). In the patent application referred toabove, it is disclosed that mature human erythrocytes expressinggalactose are preferentially attached to the surface of the substratecovered with the glycoconjugate polymer including galactose, via alectin (Allo-A) which recognizes galactose.

The present inventors have now thoroughly explored a control method forblood cell immobilization on a solid support covered with glycoconjugatepolymers via lectins, and have discovered that by means of theincubation temperature or the amount of lectins added, a specific systemof interactions among the cells and/or the carbohydrate moieties in thepolymers and the lectins can be produced; the present invention wasarrived at on this basis.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method by whichdesired immature blood cells or differentiated mature cells may beselectively, and with high yield, separated, concentrated, andrecovered, using lectins, and to provide a separation apparatusemploying this method.

This object can be accomplished by a method for selectively separatingand recovering hematopoietic cells and/or erythroblasts from a bloodsample containing differentiated mature cells, immature hematopoieticcells and erythroblasts, characterized in that the method comprises thefollowing steps:

-   (1) a step for causing said sample to interact with lectins to form    cell-lectin complexes/non-complexes under conditions in which the    cells are rendered inactive,-   (2) a step for incubating a sample containing said cell-lectin    complexes/non-complexes under said conditions with a substrate, the    surface of which is covered with a synthetic glycoconjugate polymer    having carbohydrate moieties specifically recognized by said    lectins, and immobilizing said cells on the surface of said    substrate via lectins, and-   (3) a step for separating the liquid phase from the solid phase, and    recovering desired blood cells from said liquid phase and/or said    solid phase; and optionally-   (4) a step for accelerating and stabilizing the immobilization of    cells by centrifuging said substrate and cells simultaneously prior    to or after the incubation or a step for stabilizing the    immobilization of cells by centrifuging the substrate on which the    cells are immobilized during the recovering step,-   and in that said lectins are present in an amount such that they    bind to the cells recovered from said solid phase and immobilize    these cells on the surface of the substrate, but do not immobilize    the cells recovered from said liquid phase to the surface of said    substrate.

In the method for selectively separating, the conditions under which thecells are rendered inactive may be low temperature conditions of 0° C.or above but less than 37° C., or conditions in which a pharmaceuticalagent is added which suspends cellular respiration. Furthermore, byadjusting the lectin concentration or incubation time, it becomespossible to obtain high-level selective separation which has not beenaccomplished until now.

Furthermore, the present invention also provides an apparatus for use inthe above-described selectively separating method.

The separating method and apparatus of the present invention are basedon cell-lectin and glycoconjugate-lectin interactions, and make itpossible to selectively separate cells, in particular specific cellshaving clinically important significance such as hematopoietic cells orNRBC.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a systematic diagram showing the state of hematopoietic stemcell differentiation.

FIG. 2 is a graph showing the results of the selective concentration oferythroblasts by means of changes in the lectin concentration in Example6.

DETAILED DESCRIPTIONS FOR PREFERRED EMBODIMENTS

Hereinbelow, the present invention will be discussed in detail.

In the separation method of the present invention, as a first stage, asample containing cells which are to be immobilized in a solid phase iscaused to interact with lectins which recognize carbohydrates expressedon these cells, and cell-lectin complexes/non-complexes are formed.Here, what is meant by cell-lectin complexes/non-complexes is acoexistent state in which both complexes in which cells and lectins arebound to one another, and free and unbound cells and lectins(non-complexes) are present. This step is conducted under conditionssuch that the cells are rendered inactive.

Here, what is meant by conditions in which the cells are inactive areconditions in which the mobility of the cell membranes and theself-adhesiveness thereof are lowered, and such conditions are typicallyachieved by adjusting the temperatures to within a range of from 0° C.to less than 37° C., preferably within a range of 0-36° C., morepreferably within a range of 4-30° C., and most preferably within arange of 4-22° C. However, these conditions are not necessarily limitedto the low temperature adjustment described above; such conditions mayalso be achieved by, for example, adding a pharmaceutical agent whichsuspends cellular respiration at 37° C., such as sodium azide or thelike.

The lectins are employed to recognize carbohydrates expressed on cells,which are to be immobilized. For example, as is shown in FIG. 1, whenmature leukocytes, platelets, or erythrocytes of the peripheral bloodwhich express galactose or glucose are to be immobilized, then lectinswhich recognize galactose, such as SBA, PNA, ECL, Allo A, VAA, or thelike, or lectins which recognize glucose such as Con A, LcH, PSA, or thelike, are selected. When cells expressing mannose are to be immobilized,then lectins which recognize mannose, such as LCA, GNA, CPA, or thelike, are selected.

The amount of such lectins added varies based on the type of cells whichare to be immobilized; fundamentally, the amount should be such that, inthe following second stage incubation, the cells which are to beimmobilized (the cells which are later to be recovered from the solidphase) are bonded to the surface of the substrate covered with thepolymers, while the cells which are not to be immobilized (the cellswhich are later to be recovered from the liquid phase) are not bonded tothe surface of the substrate. By means of specifying the amount oflectin added, it is possible to control, for example, the selectivitywith respect to the maturity of the cells such as leukocytes or thelike, or the selectivity among cell types such as leukocytes,erythrocytes, and the like. Concretely, the concentration is adjusted to20 mg/ml or less with respect to one cell or more. This amount addedvaries based on the type of lectins as well, so that for example, whenSBA is employed as the lectin, an amount of 5 mg/ml is sufficient.

In particular, when a polymer including a lactose structure, which has aβ-bond galactose terminal, or a mellibiose structure, which has anα-bond galactose terminal, is employed as the glycoconjugate polymer,and when CD34-negative mature cells are to be predominantly immobilizedwith controlling the immobilization of immature CD34-positive cells, theconcentration of lectin added with respect to a sample containing 2×10⁶cells may be within a range of 0.001-0.9 mg/ml, preferably within arange of 0.002-0.1 mg/ml, and more preferably within a range of0.025-0.05 mg/ml. Furthermore, when red blood cell (NRBC) are to beselectively immobilized, and other cells such as leukocytes or the likeare not to be immobilized, then the concentration of lectin with respectto a sample containing 2×10⁶ cells may be within a range of 0.001-0.3mg/ml, preferably within a range of 0.002-0.05 mg/ml, and morepreferably within a range of 0.004-0.025 mg/ml.

The incubation period in the first stage is not particularly restricted;it should be set so that the cells and the lectins interact prior to thefollowing second stage, and form cell-lectin complexes/non-complexes,and typically this will be within a range of 0-120 minutes, preferablywithin a range of 0-90 minutes, and more preferably within a range of0-60 minutes. Here, what is meant by “0 minutes” is a transfer to thesecond stage immediately after conducting the first stage. As a result,the cells which are to be immobilized and the lectins form cell-lectincomplexes/non-complexes.

Next, as a second stage in the separation method of the presentinvention, the sample containing the cell-lectin complexes/non-complexesdescribed above is incubated, under ail conditions in which the cellsare inactive, on a substrate, the surface of which is covered with theglycoconjugate polymers having carbohydrates which are specificallyrecognized by the lectins

The substrate employed here may be selected from substratesconventionally employed for cell cultivation, such as dishes, flasks,plates, cuvettes, films, fibers, beads, separable chamber slides, or thelike; it is possible to use substrates having a variety of shapesdepending on the use.

This substrate may be made of inorganic materials such as glass, silica,or the like, or organic materials such as polystyrene, polycarbonate,polysulfone, polyurethane, vinyl copolymers, or the like, as well ascomposite materials formed therefrom; however, a material which isresistant to the degree of heat required for sterilization and which iswater-resistant is preferably used. In particular, synthetic polymericmaterials are preferable from the point of view of cost and moldability,and hydrophilic materials are preferable from the point of view of thecovering effectively of the glycoconjugate polymers. When, for example,a glycoconjugate polymer containing a main chain made of polystyrene orits derivatives is employed, the use of a substrate material havingpolystyrene or its derivatives is preferable for the cover.

The surface of the substrate described above is covered with theglycoconjugate polymers having carbohydrate moieties, which arespecifically recognized by the lectins used in the first stage.

Examples of such polymers include the following:

Poly(N-p-vinyl benzyl-[O-β-D-galactopyranosyl-(1→4)-D-gluconamide])having β-galactose residues obtained by polymerizing monomerssynthesized from p-amino methyl styrene and lactose (referred to asPVLA);

Poly(N-p-vinyl benzyl-[O-α-D-glucopyranosyl-(1→4)-D-gluconamide]) havingglucose residues obtained by polymerizing monomers synthesized fromp-amino methyl styrene and maltose (referred to as PVMA);

Poly(N-p-vinyl benzyl-[O-β-D-mannopyranosyl-(1→4)-D-mannamide]) havingmannose residues obtained by polymerizing monomers synthesized fromp-amino methyl styrene and mannobiose (referred to as PVMan);

Poly(N-p-vinyl benzyl-[O-α-D-galactopyranosyl-1α→6)-D-gluconamide])having α-galactose residues obtained by the polymerization of monomerssynthesized from p-amino methyl styrene andO-α-D-galactopyranosyl-(1→6)-D-glucose (referred to as PVMeA);

Poly(N-p-vinylbenzyl-[O-6-carboxymethyl-β-D-galactopyranosyl-(1→4)-O-D-6-carboxymethyl-gluconamide])having carboxymethylated-β-galactose residues obtained by thecarboxymethylation of PVLA which is obtained by the polymerization ofmonomers synthesized from p-amino methyl styrene and lactose (referredto as PVLACOOH);

Poly(3-O-4′-vinyl benzyl-D-glucose) having glucose residues obtained bythe polymerization of monomers synthesized from p-chloromethyl styreneand glucose (referred to as PVG);

Poly(N-p-vinylbenzyl-[O-2-acetamide-2-deoxy-β-D-glucopyranosyl-(1→4)-O-D-2-acetamide-2-deoxy-β-D-glucopyranosyl-(1→4)-O-D-2-acetamide-2-deoxy-β-D-gluconamide]),poly(N-p-vinylbenzyl-[O-D-2-acetamide-2-deoxy-β-D-glucopyranosyl-(1→4)-O-D-2-acetamide-2-deoxy-β-D-gluconamide])and mixtures thereof having N-acetylglucosamine residues obtained by thepolymerization of monomers synthesized from p-chloromethyl styrene andN-acetylglucosamine (all termed PVGlcNac); and

Poly(N-p-vinyl benzyl-[O-β-D-glucopyranosyl-(1→3)-D-gluconamide]) havingβ1→3 glucose residues obtained by polymerizing monomers synthesized fromp-amino methyl styrene and laminaribiose (termed PVLam).

These glycoconjugate polymers may be homopolymers as described above ormay be copolymers with other monomers. For example, copolymers withmonomers having azide groups, which are photoreactive functional groups,are preferable in that they facilitate the formation of a covalent bondwith the substrate surface by means of the application of light.Examples of glycoconjugate polymers into which azide groups areintroduced include: poly(3-azide styrene-co-{N-p-vinylbenzyl-[O-β-D-galactopyranosyl-(1→4)-D-gluconamide]}), which is acopolymer with the PVLA described above (termed AZ-PVLA); andpoly(3-azide styrene-co-{N-p-vinylbenzyl-[O-α-D-glucopyranosyl-(1→4)-D-gluconamide]}), which is acopolymer with the PVMA described above (referred to as AZ-PVMA), andthe like.

When the incubation in the second stage is conducted in a dish or flaskused for cell culturing under the conditions same as described for thefirst stage above, then the incubation period is typically within arange of 10-120 minutes, preferably within a range of 20-90 minutes, andmore preferably within a range of 30-70 minutes. As a result, thecell-lectin complexes to be immobilized are attached to the substratevia the glycoconjugate polymers on the surface of the substrate.

On the other hand, under conditions in which the cell precipitationperiod can be ignored in a column filled with beads or non-woven fabric,this period may be further shortened. The shortening of the period canalso be attained by acceleration and stabilization of the precipitationand immobilization of cells by means of centrifuging the substrate andcells simultaneously prior to or after the incubation of the secondstage. The centrifuging period is sufficient within a range of 30minutes or less, and it preferably be 10 minutes or less from the pointof view of shortening time. The centrifuging force will vary dependingon the type of cells to be immobilized or types or concentrations oflectins used, and in general it preferably be in a range of 30-450 G.

Next, as a third stage of the separation method of the presentinvention, the solid phase which is immobilized on the substrate, andthe liquid phase which remains unimmobilized are separated. In the casein which a column filled with beads or plates covered with thecarbohydrates is employed, a sample containing the cell-lectincomplexes/non-complexes obtained in the first stage may simply beintroduced into the column, and thereby, it is possible to separate thesolid phase by recovering the liquid phase from the outlet of thecolumn.

When chromosomes of the cells attached on a substrate, such as a chamberslide or dish, are examined, the liquid phase staying unattached in theabove recovering step is removed, and the substrate on which cells areattached is then centrifuged to stabilize the immobilization of thecells which results in a uniform immobilized image. The centrifugingperiod is sufficient within a range of 30 minutes or less, and itpreferably be 10 minutes or less from the point of view of shorteningtime. The centrifuging force will vary depending on the type of cells tobe immobilized or types or concentrations of lectins used, and ingeneral it is preferably 30 G or more, more preferably in a range of100-400 G, and further preferably in a range up to 1,000 G.

More specifically, in the case in which, for example, PVLA is employedas the glycoconjugate polymer, and SBA or the like is employed as thegalactose-specific lectin in order to recover desired hematopoieticcells from liquid and/or solid phase, then the ease of attachment goesin the order of mature erythrocytes, NRBC>leukocytes>immatureCD34-positive cells. Accordingly, if the amount of lectin is reduced,the CD34-positive cells will first become unattached, and then theleukocytes will become unattached, and finally, the mature erythrocytesand NRBC will be the only cells which are selectively attached. In otherwords, if to the separation method of the present invention is conductedusing an amount of added lectin such that only CD34-positive cellsremain unattached, then the CD34-positive cells will be selectivelycontained in the liquid phase, and it will be possible to recover theseCD34-positive cells with a high degree of purity. Furthermore, if theamount of added lectin is further reduced, and only mature erythrocytesand NRBCs are attached, then it is possible to selectively recovermature erythrocytes and NRBCs from the solid phase.

In this separation method of the present invention, it is also possibleto remove granulocytes and leukocytes and the like from the blood sampleobtained in advance prior to the first stage, so as to concentrate thedesired cells. In such a case, multi-purpose high-density liquid set tospecific gravity of about 1.077 such as Ficoll Paque, Histo Paque,Percall, or the like may be generally used, while in the presentinvention, those having a specific gravity of 1.085-1.10 areparticularly used in a pretreatment for separating and concentratingNRBC from a blood sample. In practice, the present inventors confirmedthat when a high-density liquid, such as Ficoll Paque, Histo Paque,Percall, or the like, having a specific gravity of 1.095 is used, theresulting recovered amounts of NRBC via lectin were unproved about 1.5times larger than those obtained using a conventional high-densityliquid having a specific gravity of 1.077. Although many investigatorshave considered the effects of these pretreatments for a long time, noconclusion has been obtained due to variations among individuals and thelike. Therefore, the other conditions may also be appropriately employedin the selective separating method of the present invention.

Furthermore, as shown in FIG. 1, it is also of course possible toconduct the method in the same way using synthetic glycoconjugatepolymer of glucose family and lectin.

In general, negative separation, in which cells other than those whichare desired are immobilized, and positive separation, in which the cellswhich are desired are immobilized and concentrated, are known as methodsfor separating cells; however, the separation method of the presentinvention makes use of both negative and positive separation byappropriately adjusting the concentration of lectins.

In the selective separation of hematopoietic stem cells which arepresent in very small amounts, in order to reduce the number of thecells which are wasted, the method described above may be repeated anumber of times to make it possible to increase the yield of the desiredcells.

The blood sample which is separated and refined by means of theseparation method of the present invention may be from any source,including peripheral blood; however, in the selective recovery of stemcells, bone marrow fluid, umbilical blood, or placental blood ispreferable. Furthermore, in the selective recovery of NRBCs, umbilicalblood or maternal blood is preferable.

EXAMPLES

Hereinbelow, a case will be concretely discussed in which, following theseparation method of the present invention, and employing AZ-PVLA as aglycoconjugate polymer, and using a phosphate-buffered physiologicalsaline (PBS) supplemented with 0.1% by weight of bovine serum albumin asa cell suspension. In this case, immature hematopoietic stem cells areselectively separated and recovered.

1: Incubation Conditions Example 1

Temperature Effects (1)

First stage: Cord blood mononucleated cells monocytes were obtained ascells treated with ammonium chloride after centrifugation on FicollPaque. In a tube made by polypropylene, PBS containing a variety ofconcentrations of SBA (lectin specific for galactose) was added to asuspension of the mononucleated cells derived from cord blood of 2×10⁶cells per ml, and the mixture was incubated at a temperature of 4° C.for a period of 30 minutes and was gently stirred at intervals of 5minutes.

Second stage: after the completion of the incubation described above,the suspension was transferred to a dish having a diameter of 35 mmwhich was coated with AZ-PVLA, the tube was further rinsed with 1 ml ofthe isotonic salt solution described above, and this rinse liquid wasalso added to the dish, and incubation was conducted at a variety oftemperatures from 4° C. to 37° C. for a period of 60 minutes.Alternatively, the dish and cells were centrifuged for a predeterminedperiod at 90 G in place of the incubation for 60 minutes, or thesubstrate and cells were incubated for 15 minutes and then centrifugedfor a predetermined period in place of the incubation for 60 minutes. Inaddition, both of the centrifuging treatments were conductedconcomitantly.

Third stage: after stirring, the suspension liquid was recovered,washing was conducted with 1 ml of the PBS, and the solid phase (dish)and liquid phase (suspended liquid) were separated.

The recovered cell count in the cellular suspension liquid obtained wasmeasured using an automated blood cell counter, and the proportion(attachment ratio) of attaching cells with respect to the number ofcells used was calculated. As a result, it was discovered that there wasa trend for the attachment ratio to increase as the amount of lectinadded increased at all temperatures. The amount of added lectin (SBA),which was minimally necessary in order to cause the adhesion of 80% ofthe mononucleated cells at each temperature, was as shown in Table 1below.

TABLE 1 Minimum Amount of Added SBA Required to Cause 80% of ProcessingMononucleated cell Attachment Temperature to applying 2 × 10⁶ cells 37°C. 1.0 mg 30° C. 0.5 mg 10° C. 0.05 mg  4° C. 0.025 mg

From these results, it can be seen that the amount of added lectin (SBA)necessary to cause the attachment of 80% of the mononucleated cellsdecreased along with a decrease in temperature, so that in other words,by reducing the incubation temperature, the attachment efficiency couldbe increased, so that attachment and separation became possible withsmall amounts of lectin. Under the processing temperature at 40° C., acellular attachment of approximately 50% was observed at 0.01 mg. Thefact that this cellular attachment was specific for carbohydrate vialectin was confirmed by the fact that, by adding the galactose solutionin various concentrations to the dish, the cellular adhesion wasinhibited by 60-90% both at 4° C. and 37° C.

In addition, in the case in which centrifuging at 90 G was conducted inplace of the incubation for 60 minutes, centrifuging for not less than 3minutes provided the same cell-attachment as that obtained by theincubation for 60 minutes. Furthermore, in the case in which incubationfor 15 minutes followed by centrifuging at 90 G was conducted in placeof the incubation for 60 minutes, stable cell-attachment was obtained bycentrifuging for not less than 2 minutes. These centrifuging treatmentswere able to facilitate and stabilize the selective attachment of thecells via lectins and contributed to shortening processing period,unless such centrifuging was so excessive to destroy the cells resultingin unselective adhesion.

Example 2

Temperature Effects (2)

A procedure was followed under conditions which were identical to thoseof Example 1, with the exception that the lectins employed were PNA andECL (both of which are galactose-specific), and the results shown inTable 2 below were obtained.

TABLE 2 Amount of Cellular Attachment Ratio Lectin Added ProcessingProcessing Lectin (mg/2 × 10⁶ cells) Temperature 4° C. Temperature 37°C. PNA 0.72 73 44 ECL 0.02 81 58

From the results in Table 2, it became clear that, irrespective of thetype of lectin employed, a relationship was present between the cellularattachment ratio and the incubation temperature, as shown above.

Example 3

Contents of Temperature Effects

The same type of experiment was conducted under conditions identical tothose of the incubation at 37° C. in Example 1, and sodium azide wasadded to the cell suspension. The results are shown in Table 3. Here,the attachment of the cells is expressed in terms of the proportion ofcells recovered which were not attached (the recovery ratio).

TABLE 3 Incubation Conditions Recovery (Amount of SBA Added: 1 mg/2 ×10⁶ cells) Ratio (%) Incubation temperature of 37° C. 14.8 Incubationtemperature of 37° C. + 3.9 10 mM sodium azide Incubation Temperature of4° C. 6.7

From the results shown above, it can be seen that the cellularattachment via lectin increases with a decrease in temperature; however,this phenomenon is also observed if sodium azide, which is known tosuppress metabolic activities, is added even if the temperature is notreduced. That is to say, the temperature-dependent affinity of thelectin for the cells is affected by the cells affected by the cellularmembrane mobility, and a tendency is observed for the affinity toincrease as the membrane mobility decreases.

2. Selective Affinity Example 4

Selective Affinity Based on the Maturity of Leukocytes

Immature leukocytes express a surface marker termed CD34 on the surfacethereof (they are CD34-positive) and are known to become CD34-negativeas they mature. Conventionally, the selective immobilizing of immaturecells was conducted using CD34 antibodies. Here, on solid surfaces withPVLA having a β-bond galactose terminus attached (at incubationtemperatures of 4° C. and 37° C.), and PVMeA having an α-bond galactoseterminus attached (at an incubation temperature of 4° C.), selectiveattachment was investigated using various amounts of added lectin. Theresults thereof are shown in Table 4. The described values of SBA in thetable represent the amount added with respect to 2×10⁶ cells

TABLE 4 Cellular Attachment Ratio (%) Leukocyte SBA 0.025 mg SBA 0.05 mgSBA 1 mg Maturity PVMeA (4° C.) PVLA (4° C.) PVLA (37° C.) Immature,CD34- 20 15 12 positive cells Mature, CD34- 76 73 70 negative cells

From the results above, it was discovered that by means of theseparation method of the present invention using lectin, it is possibleto selectively separate CD34-positive immature cells and CD34-negativemature cells. Moreover, by means of setting the incubation temperatureto a low temperature, the amount of added lectin required was reduced toapproximately 1/20 of that formerly required. Furthermore, when PVMeAwas employed as the glycoconjugate polymer, in comparison with the casein which PVLA was employed, selective attachment was obtained even whenthe amount of added lectin was further reduced.

It is generally known that SBA has a stronger affinity to galactose ofα-bond type. Therefore, the results, in which the selective cellattachment to PVMeA having α-bond type galactose terminal was observedat lower lectin concentration, demonstrate that the separating method ofthe present inventors is based on quite specific affinity of lectins.Furthermore, the present method clarified that thelow-temperature-incubation is an effective process which can stronglyenforce the affinity of lectins.

Example 5

Selective Affinity among Blood Cells

An experiment was conducted which was identical to that of Example 1 andthe incubation temperature was set at 4° C., and the amount of addedlectin required to cause the attachment of 95% of erythrocytes and ofleukocytes derived from umbilical cord blood was determined. In thiscase, the hemolysis was not conducted. The results thereof are shown inTable 5 below.

TABLE 5 Amount of Added Lectin Required to Cause 95% or more ofErythrocytes Cells or Leukocytes to Attach (2 × 10⁶ cells) Leukocytes300 micrograms or more Erythrocytes  50 micrograms or more

As shown in Table 5, in comparing the attachment via lectin of theerythrocytes and leukocytes, the erythrocytes showed higher affinity,and were capable of attachment at lower levels of added lectin.

Example 6

Separation of Umbilical Cord Blood Erythroblasts

The incubation temperature was set to 4° C., and the amount of lectin(SBA) added was altered to a very small amount of 50 micrograms or lessper 2×10⁶ cells, and the cells attaching to a dish covered with PVLAwere investigated as in Example 5. In addition, the results obtained bycentrifuging the dish and attached cells at 450 G after separating thesolid phase from liquid phase in the third step were compared.

The method employed in this investigation was such that the attachingcells were allowed to dry on the dish, cells were then stained withhematoxylin and erythrosin, and positively stained erythroblasts werecounted, and 100 cells in a randomly selected area were counted and thenumber of erythroblasts contained therein was evaluated. The resultsthereof are shown in FIG. 2. In the figure, PRE indicates a comparativeexample in which a large amount (300 micrograms) of lectin was added andalmost all cells were caused to attach.

If the amount of lectin added is decreased, and the attachment ofleukocytes which have a lower affinity for the lectin is preferentiallyreduced, then mature erythrocytes and erythroblasts are selectivelycaused to attach to the dish. Accordingly, it is possible to detecterythroblasts which are present at low levels in umbilical cord blood,at a high probability of 1 or more/100 cells. Furthermore, whencentrifuging at 450 G for not less than 3 minutes was conducted afterseparating the solid phase from liquid phase, the attached cellsexhibited uniform spherical shapes which provided good stainingsensitivity and therefore made it easy to visually recognizeerythroblasts with a microscope. However, when the centrifugingtreatment was not sufficient (for example, in a case whereincentrifuging force was too small or centrifuging period was too short),uniform cell staining image could not be obtained which resulted indifficulties in visual recognition of desired cells with a microscope.Accordingly, it was found that it is very important to centrifuging thecells attached to the substrate with appropriate conditions when oneintends to improve the efficacy of a cytological examination, whichrequires detection of fine nucleus structure by cell staining.

Example 7

Concentration of Fetal Erythroblasts in the Maternal Blood

Following the method of Example 6, a cell fraction separated by theFicoll Paque was recovered from maternal blood, 10 micrograms of alectin (SBA) was added with respect to 2×10⁶ cells, and this wasincubated for a period of 30 minutes at a temperature of 18° C. in aPVMeA-covered separable slide chamber. For the purposes of comparison, acase was also evaluated in which the amount of lectin added was 300micrograms. The experimental results for 20 examples are shown in Table6 below.

TABLE 6 Number of Positively Amount of Stained Erythroblasts LectinAdded Adhered to the Dish 10 micrograms 300 micrograms 0-1 1 instance 19instances* 2-5 3 instances 1 instance*  6-10 7 instances — 11-30 8instances — 31 or more 1 instance —

When 300 micrograms was added, too many nucleated leukocytes other thanerythroblasts were caused to attach, so that a miss count occurred.

In consideration with the above results, the optimal amounts of lectin(SBA) to be added for detecting fetal erythroblasts were estimated. Theresults estimated from 20 instances are indicated in the following Table7. In each case, a maternal body was examined based on informed consentby echo imaging to ascertain that she carries healthy boy. Theerythroblasts separated from the collected maternal blood were examinedby FISH assay using Aneu Vysion Assay Kit (VYSIS, INC.) to detect aY-probe.

TABLE 7 The Amount of Lectin Added (μg) 2 4 8 12 16 The Number of NRBCdetected 0 0.16 1.00 1.07 0.97 (relative value when the number of NRBCdetected with 8 μg of lectin is assumed as 1.00)

These results show that when the amount of lectin added is decreased andthe attachment of leukocytes is reduced, it is possible to selectivelyaccumulate erythroblasts, and it is possible to efficiently detecterythroblasts, which are useful in genetic diagnosis, from maternalblood. In addition, it was found that there was an apparent lower-limitof the amount of lectin to be added, and that the loss of erythroblastswas reduced when 8 μg or more of lectin was added. In such cases,contamination with nucleated cells or leukocyte was gradually increasedwhen the amount of lectin exceeded 20 μg, the contamination occurred soabundantly that the recognition of NRBC became difficult when the amountof lectin reached to 32 μg or more. Furthermore, these results werereproducible when PVLA was employed as the glycoconjugate polymer.

On the other hand, with the maternal blood samples containing smallamounts of erythrocyte components such as erythroblasts, gooderythrocyte selective attachment was reproduced without the incubationwith lectin at the first stage.

Furthermore, 8 samples of maternal bodies who carries a boy wereexamined at this time, and Y-probes specific for boy were detected inthe 8 samples which means that fetal cells can be recovered frommaternal blood with high yield. Therefore, it was found that theseparation method for nucleated erythroblasts using a lectin of theinvention is an effective means for detecting fetal cells from maternalblood with no- or low-invasion and examining the fatal chromosomes.

The fractions of CD34-positive cells concentrated in accordance withExample 4 were recovered, and their colony-forming abilities werecompared using commercially available assay kit (MethoCult GF H4434,Stem Cell Technologies Inc.). As a result, the CD34-positive cellsconcentrated by the separation method via lectin of the inventionexhibited colony-forming abilities of 8.8 times larger than thatobtained without such separation. These results demonstrate that thetreatment with lectin can effectively increase the hematopoietic cellswithout affecting their subsequent colony-forming abilities.Accordingly, it is believed that the cell separating method of theinvention can provide a transplant graft with reduced lymphocyte, whichcan alleviate GVHD, to a patient in need of stem cell transplant, andtherefore, the separating method of the invention can be used ineffective detection of oncogenes derived in each stage ofdifferentiation of leukemia cells.

Industrial Applicability

As described in detail above, the separation method and separationapparatus of the present invention are based on interaction amongcells-lectins and glycoconjugate polymers-lectins. In greater detail,the present invention utilizes the fact that the changes in attachmentproperties depending on the state of activity of cells or the amount oflectin added, and changes in attachment selectivity depending of thetype of cells were observed in the above interactions. By employing theseparation method and separation apparatus of the present invention, itis possible to selectively separate and recover cells which have greatclinical significance, such as hematopoietic stem cells or NRBCs.

1. A method for selectively separating and recovering hematopoieticcells and/or erythroblasts from a blood sample containing differentiatedmature cells, immature hematopoietic cells and erythroblasts,characterized in that the method comprises the following steps: (1) astep for causing said sample to interact with lectins to formcell-lectin complexes/non-complexes under conditions in which the cellsare rendered inactive, (2) a step for incubating a sample containingsaid cell-lectin complexes/non-complexes under said conditions with asubstrate, the surface of which is covered with a syntheticglycoconjugate polymer having carbohydrate moieties specificallyrecognized by said lectins, and immobilizing said cells on the surfaceof said substrate via lectins, and (3) a step for separating the liquidphase from the solid phase, and recovering desired blood cells from saidliquid phase and/or said solid phase; and in that said lectins arepresent in an amount such that they bind to the cells recovered fromsaid solid phase and immobilize these cells on the surface of thesubstrate, but do not immobilize the cells recovered from said liquidphase to the surface of said substrate.
 2. A method according to claim1, characterized in that said method further comprises: (4) a step foraccelerating and stabilizing the immobilization of cells by centrifugingsaid substrate and cells simultaneously prior to or after the incubationor a step for stabilizing the immobilization of cells by centrifugingthe substrate on which the cells are immobilized during the recoveringstep.
 3. A method according to claim 1 or 2, characterized in that saidconditions under which said cells are rendered inactive are: lowtemperature conditions of 0° C. or above but less that 37° C., orconditions in which a pharmaceutical agent is added which suspendscellular respiration.
 4. A method according to claim 1 or 2,characterized in that the concentration of said lectins is within arange of 20 mg/ml or less per cell.
 5. A method according to claim 1 or2, characterized in that the incubation period of step (1) is set withina range of 0-120 minutes, and the incubation period of step (2) is setto a range of 10-120 minutes.
 6. A method according to claim 1,characterized in that said substrate is selected from a group consistingof dishes, flasks, plates, cuvettes, films, fibers, or beads made ofglass, polystyrene, polycarbonate, polysulfone, polyurethane, or vinylcopolymer.